Naoki Yamamoto

Background and Objectives: Posterior chamber phakic implantable contact lenses (Phakic-ICL) are widely used for refractive correction due to their efficacy and safety, including minimal corneal endothelial cell loss. The Collamer-based EVO+ Visian implantable contact lens (ICL), manufactured from Collamer, which is a blend of collagen and hydroxyethyl methacrylate (HEMA), has demonstrated excellent long-term biocompatibility and optical clarity. Recently, hydrophilic acrylic Phakic-ICLs, such as the Implantable Phakic Contact Lens (IPCL), have been introduced. This study investigated the material differences among Phakic-ICLs and their interaction with fibronectin (FN), which has been reported to adhere to intraocular lens (IOL) surfaces following implantation. The aim was to compare Collamer, IPCL, and LENTIS lenses (used as control) in terms of FN distribution and cell adhesion using a small number of explanted Phakic-ICLs. Materials and Methods: Three lens types were analyzed: a Collamer Phakic-ICL (EVO+ Visian ICL), a hydrophilic acrylic IPCL, and a hydrophilic acrylic phakic-IOL (LENTIS). FN distribution and cell adhesion were evaluated across different regions of each lens. An in vitro FN-coating experiment was conducted to assess its effect on cell adhesion. Results: All lenses demonstrated minimal FN deposition and cellular adhesion in the central optical zone. A thin FN film was observed on the haptics of Collamer lenses, while FN adhesion was weaker or absent on IPCL and LENTIS surfaces. Following FN coating, Collamer lenses supported more uniform FN film formation; however, this did not significantly enhance cell adhesion. Conclusions: Collamer, which contains collagen, promotes FN film formation. Although FN film formation was enhanced, the low cell-adhesive properties of HEMA resulted in minimal cell adhesion even with FN presence. This characteristic may contribute to the long-term transparency and biocompatibility observed clinically. In contrast, hydrophilic acrylic materials used in IPCL and LENTIS demonstrated limited FN interaction. These material differences may influence extracellular matrix protein deposition and biocompatibility in clinical settings, warranting further investigation.

Developmental toxicity testing is essential to identify substances that may harm embryonic development. This study aimed to establish a protocol for evaluating developmental toxicity using human induced pluripotent stem cells (iPSCs) by analyzing cellular activity and gene expression changes. Two ICH S5(R3) positive substances, valproic acid (VPA), which is a substance previously detected as positive by other test methods, and thalidomide (Thalido), were examined during early trichoderm differentiation without fetal bovine serum. RNA-seq analysis identified seven candidate genes, including TP63, associated with altered expression following exposure to VPA or Thalido. These genes were implicated in pathways related to tissue development, cell growth, and molecular interactions. While the assay effectively detected VPA and Thalido, its limitations include testing only soluble substances and focusing on early differentiation stages. Nevertheless, the protocol demonstrates potential for the classification and evaluation of emerging modality drugs based on physical properties such as solubility, polarity, and pH. Integration with AI analysis may enhance its capacity to uncover genetic variations and evaluate previously uncharacterized substances. This study provides a foundation for alternative developmental toxicity testing methods, with further refinements in the culture method expected to improve accuracy and applicability in regulatory toxicology.

Various tips are available for phacoemulsification in cataract surgery. Evidence-based data can inform ophthalmologists, especially inexperienced ones, on tip selection. We retrospectively evaluated the energy efficiency and other parameters of two ultrasonic phacoemulsification and aspiration tips across different nuclear hardness grades in 342 cataract patients (342 eyes) with nuclear hardness grades II to IV. Surgical procedures, ultrasound settings, and instrumentation were standardized. All surgeries were performed by one experienced doctor. We compared the metal INTREPID® Balanced Tip (M-tip) with the INTREPID® Hybrid Tip (P-tip), which has a polymer coating. The M-tip required significantly less total ultrasound time and cumulative dissipated energy (CDE) than the P-tip for grades III and IV, while the P-tip had a shorter aspiration time and less estimated fluid aspirated for grade II. No differences in corneal endothelial cell loss were observed. Lens rupture rates were low: 0.47% for the M-tip and 0.78% for the P-tip. Multiple regression analysis showed that CDE increased with nuclear hardness. These findings suggest that the M-tip is efficient for harder lenses, while the P-tip may be advantageous for softer lenses, informing optimal tip selection in cataract surgery. Further research is suggested to elucidate their clinical significance.

The prevalence of presbyopia and nuclear cataracts (NUC) is reported to be higher in tropical areas than that in other regions, suggesting a potential influence of high temperatures on lens health. Transient receptor potential vanilloid (TRPV) channels play a crucial role in detecting ambient temperatures across various species, with TRPV1 and TRPV4 expressed in lens epithelial cells. In this study, we investigated whether ambient temperatures affect TRPV1 and TRPV4 activity in the lens, potentially contributing to the development of presbyopia and NUC. We conducted experiments using cultured human lens epithelial cell lines under different temperature conditions. Our results revealed that the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and p38 pathways, downstream molecules of TRPV1, were activated, while Src family kinase, a downstream molecule of TRPV4, was inhibited at 37.5 °C culture compared to 35.0 °C. Confocal microscope images demonstrated higher expression of TRPV1 in 3D-structured cells under high-temperature culture conditions. Additionally, in organ culture lenses, higher elasticity was observed at elevated temperatures compared to that at lower temperatures. These results suggest that high ambient temperatures may induce lens sclerosis via TRPV1 activation, potentially contributing to the development of presbyopia and NUC.

Purpose: To explore lens capsule pathological characteristics in intraocular lens (IOL) dislocation after cataract surgery in patients with atopic dermatitis (AD). Setting: University hospital department of ophthalmology. Design: Case series with clinicopathological correlations. Methods: Lens capsules and surrounding tissues excised during surgery from eyes with AD (AD group) and eyes without AD (non-AD group) with IOL dislocation were histologically evaluated. Hematoxylin and eosin staining was used to assess abnormal changes in lens epithelial cells (LECs). Masson trichrome staining distinguished the fibrous metaplasia around the lens capsule into high-density and low-density fibrosis. Capsular splitting (thinning) was identified in both stained preparations. Results: The IOL dislocation morphology in the AD group (10 eyes of 10 patients) included 7 cases of capsular bag dislocation (CBD) and 3 cases of dead bag syndrome (DBS), with an average duration to IOL dislocation of 11.5 ± 5.6 years. All patients in the non-AD group (12 eyes of 12 patients) had CBD, averaging 10.2 ± 5.7 years to dislocation. Abnormal LECs, low-density fibrosis, and capsular splitting were observed in 9 (90), 9 (90), and 6 (60) of the patients in the AD group compared with 6 (50), 3 (25), and 2 (18), respectively, in the non-AD group (total n [%]). Conclusions: Compared with the non-AD group, the AD group exhibited higher frequencies of morphological changes in LECs, low-density fibrosis around the lens capsule, and capsular splitting characteristics of DBS. These results suggest LEC degeneration and increased lens capsule fragility occurred in patients with AD.

In a previous study, a novel human corneal-like epithelium model utilizing an immortalized human corneal epithelial cell line (iHCE-NY1) was developed as an alternative to animal models to identify chemicals not classified under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) and was evaluated following the criteria of Test Guideline 492 of the Organization for Economic Co-operation and Development (OECD). In the present study, our aim was to establish an eye irritation test protocol using the iHCE-NY1 model to classify liquid chemicals under the GHS ocular hazard categories: no effect, no classification (No Cat.), Category 2 (Cat. 2) reversible effects, and Category 1 (Cat. 1) irreversible eye damage. The protocol involved exposing the iHCE-NY1 model to 31 liquid test chemicals for 5 min, followed by observation at post-incubation periods (PIPs) to assess recovery. Classification was based on cell viability, and histopathological findings on PIP days 7, 14, and 21. The outcomes were compared with an established database of classifications. All Cat. 1 liquid chemicals, 62.5% of No Cat., and 63.2% of Cat. 2 were correctly categorized. This study demonstrates that the iHCE-NY1 model can not only distinguish No Cat. test liquid chemicals but also differentiate between Cat. 2 and Cat. 1 liquid chemicals.

INTRODUCTION: Increasing numbers of cases of mild asymptomatic pulmonary alveolar proteinosis (PAP) are being reported with the recent increase in chest computed tomography (CT). Bronchoscopic diagnosis of mild PAP is challenging because of the patchy distribution of lesions, which makes it difficult to obtain sufficient biopsy samples. Additionally, the pathological findings of mild PAP, particularly those that differ from severe PAP, have not been fully elucidated. This study aimed to clarify the pathological findings of mild PAP and the usefulness of optical biopsy using probe-based confocal laser endomicroscopy (pCLE). METHODS: We performed bronchoscopic optical biopsy using pCLE and tissue biopsy in five consecutive patients with PAP (three with mild PAP and two with severe PAP). We compared the pCLE images of mild PAP with those of severe PAP by integrating clinical findings, tissue pathology, and chest computed tomography images. RESULTS: pCLE images of PAP showed giant cells with strong fluorescence, amorphous substances, and thin alveolar walls. Images of affected lesions in mild PAP were equivalent to those obtained in arbitrary lung lesions in severe cases. All three patients with mild PAP spontaneously improved or remained stable after ≥3 years of follow-up. Serum autoantibodies to granulocyte-macrophage colony-stimulating factor were detected in all five cases. CONCLUSION: Optical biopsy using pCLE can yield specific diagnostic findings, even in patients with mild PAP. pCLE images of affected areas in mild and severe PAP showed similar findings, indicating that the dysfunction level of pathogenic alveolar macrophages in affected areas is similar between both disease intensities.

BACKGROUND: Bronchial thermoplasty (BT) is a recently developed non-pharmacological therapy for refractory bronchial asthma. Although increasing evidence has suggested that BT is effective for various phenotypes of severe asthma, its safety and efficacy in patients with severe irreversible impaired lung function are unclear. OBJECTIVES: To assess the efficacy and safety of BT in patients with refractory asthma, including patients with a severely impaired forced expiratory volume in 1 second (FEV1). DESIGN: This was a single-center, retrospective, observational cohort study. METHODS: We retrospectively reviewed the medical records of 15 patients with refractory asthma (Global Initiative for Asthma step 4 or 5), including patients with severely impaired airflow limitation (% predicted pre-bronchodilator FEV1 <60%), who had undergone BT between June 2016 and January 2022. We analyzed the efficacy (change in asthma symptoms, exacerbation rate, pulmonary function, asthma medication, and serum inflammatory chemokine/cytokines before and after BT) and complications in all patients. We compared these data between patients with severe obstructive lung dysfunction [group 1(G1)] and patients with FEV1 ⩾ 60% [group 2 (G2)]. RESULTS: Six patients were in G1 and nine were in G2. Clinical characteristics, T2 inflammation, and concurrent treatment were equivalent in both groups. BT significantly improved asthma-related symptoms (measured using the Asthma Control Test and Asthma Quality of Life Questionnaire scores) in both groups. FEV1 was significantly improved in G1 but not in G2. Four patients in G2, but none in G1, experienced asthma exacerbation requiring additional systemic corticosteroids (including two requiring prolonged hospitalization) after BT. Long-term responders (patients who reduced systemic or inhaled corticosteroid without newly adding biologics in a follow-up > 2 years) of BT were identified in G1 and G2 (n = 2, 33.3% and n = 4, 44.4%, respectively). CONCLUSION: BT in patients with refractory asthma and severe airflow limitation is equally safe and efficacious as that in patients with moderate airflow limitation.

Presbyopia is caused by age-related lenticular hardening, resulting in near vision loss, and it occurs in almost every individual aged ≥50 years. The lens experiences mechanical pressure during for focal adjustment to change its thickness. As lenticular stiffening results in incomplete thickness changes, near vision is reduced, which is known as presbyopia. Piezo1 is a mechanosensitive channel that constantly senses pressure changes during the regulation of visual acuity, and changes in Piezo1 channel activity may contribute to presbyopia. However, no studies have reported on Piezo1 activation or the onset of presbyopia. To elucidate the relevance of Piezo1 activation and cross-linking in the development of presbyopia, we analysed the function of Piezo1 in the lens. The addition of Yoda1, a Piezo1 activator, induced an increase in transglutaminase 2 (TGM2) mRNA expression and activity through the extra-cellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH2-terminal kinase1/2 pathways. In ex vivo lenses, Yoda1 treatment induced γ-crystallin cross-linking via TMG2 activation. Furthermore, Yoda1 eye-drops in mice led to lenticular hardening via TGM2 induction and activation in vivo, suggesting that Yoda1-treated animals could serve as a model for presbyopia. Our findings indicate that this presbyopia-animal model could be useful for screening drugs for lens-stiffening inhibition.

BACKGROUND: Gastrointestinal injuries caused by nonsteroidal anti-inflammatory drugs (NSAIDs) is a serious side effect in patients with rheumatoid arthritis (RA). However, effective therapeutic strategies have yet to be established. In this study, we investigated the therapeutic effects of teprenone (TEP), a gastric mucosal protective drug, on NSAID-induced gastrointestinal injuries in rats with RA (AA rats). METHODS: Gastrointestinal injury was induced by oral administration of indomethacin (IMC), a typical NSAID. TEP was orally administered after IMC-induced gastrointestinal bleeding, and the stomach, jejunum, and ileum were excised. RESULTS: On day 14 of IMC administration, lesion areas in the stomach, jejunum, and ileum were significantly larger in AA rats than in normal rats. When TEP was orally administered to AA rats, the lesion areas in the stomach, jejunum, and ileum significantly decreased compared with those in control rats (IMC-induced AA rats). Therefore, we measured NOS2 mRNA and NO levels, which were significantly decreased in rats with IMC-induced AA after treatment with TEP. CONCLUSIONS: These results suggest that the oral administration of TEP may be useful for the treatment of NSAID-induced gastrointestinal injuries in patients with RA.

Regulation of ion and water microcirculation within the lens is tightly controlled through aquaporin channels and connexin junctions. However, cataracts can occur when the lens becomes cloudy. Various factors can induce cataracts, including diabetes which is a well-known cause. The most common phenotype of diabetic cataracts is a cortical and/or posterior subcapsular opacity. In addition to the three main types and two subtypes of cataracts, a vacuole formation is frequently observed; however, their origin remains unclear. In this study, we focused on the aquaporins and connexins involved in diabetes-induced cataracts and vacuoles in Nile grass type II diabetes. The results showed that the expression of aquaporin 0 and aquaporin 5 increased, and that of connexin 43 decreased in diabetic rat lenses. Additionally, aquaporin 0 and 5 were strongly localized in peripheral of vacuoles, suggesting that aquaporins are involved in vacuoles formation. Transillumination photography revealed large vacuoles at the tip of the Y-suture in the anterior capsule of the diabetic lens, and several small vacuoles were observed in the posterior capsule. Within the vacuoles, cytoplasmic degradation and aggregation of fibrous material were observed. Our findings suggest that aquaporins are potential candidate proteins for preventing vacuole formation.

Advanced glycation end products (AGEs) in lens proteins increase with aging, thus inducing cataracts and/or presbyopia. Hesperetin (Hst), which is an abundant plant flavanone largely derived from citrus species, and its derivatives attenuate cataracts and presbyopia in vivo and in vitro; however, no reports have described its effects on AGE formation in lens proteins. The present study demonstrated that AGEs in lens proteins increase with age in mice. Additionally, it showed that Hst can prevent AGEs and N(ε)‑carboxymethyl‑lysine generation and modification of lens proteins using in vitro in human lens epithelial cell lines and ex vivo in mouse lens organ cultures. Furthermore, treatment with Hst prevented lens hardening and decreased chaperone activity in lens proteins. These results suggested that Hst and its derivatives are good candidates for the prevention of presbyopia and cataracts.

Knee osteoarthritis (Knee OA) is an irreversible condition that causes bone deformity and degeneration of the articular cartilage that comprises the joints, resulting in chronic pain and movement disorders. The administration of cultured adipose-derived stem cells (ADSCs) into the knee joint cavity improves the clinical symptoms of Knee OA; however, the effect of synovial fluid (SF) filling the joint cavity on the injected ADSCs remains unclear. In this study, we investigated the effect of adding SF from Knee OA patients to cultured ADSCs prepared for therapeutic use in an environment that mimics the joint cavity. An increase in the viability of ADSCs was observed following the addition of SF. Gene expression profiling of SF-treated ADSCs using DNA microarrays revealed changes in several genes involved in cell survival. Of these genes, we focused on FOSL1, which is involved in the therapeutic effect of ADSCs and the survival and proliferation of cancer stem cells. We confirmed the upregulation of FOSL1 mRNA and protein expression using RT-PCR and western blot analysis, respectively. Next, we knocked down FOSL1 in ADSCs using siRNA and observed a decrease in cell viability, indicating the involvement of FOSL1 in the survival of ADSCs. Interestingly, in the knockdown cells, ADSC viability was also decreased by SF exposure. These results suggest that SF enhances cell viability by upregulating FOSL1 expression in ADSCs. For therapy using cultured ADSCs, the therapeutic effect of ADSCs may be further enhanced if an environment more conducive to the upregulation of FOSL1 expression in ADSCs can be established.

Enhancement of density via human lens epithelium (HLE) cell proliferation is the underlying cause of nuclear cataracts. Moreover, our previous epidemiological study demonstrated that the risk of nuclear cataract development is significantly higher under elevated environmental temperatures compared with under lower temperatures. The present study investigated the relationship between temperature and cell proliferation in terms of mitochondrial function, which is a nuclear cataract‑inducing risk factor, using two different HLE cell lines, SRA01/04 and immortalized human lens epithelial cells NY2 (iHLEC‑NY2). Cell proliferation was significantly enhanced under the high‑temperature condition (37.5˚C) in both cell lines. The cell growth levels of SRA01/04 and iHLEC‑NY2 cells cultured at 37.5˚C were 1.20‑ and 1.16‑fold those in the low‑temperature cultures (35.0˚C), respectively. Moreover, the levels of cytochrome c oxidase mRNA (mitochondrial genome, cytochrome c oxidase‑1‑3) and its activity in SRA01/04 and iHLEC‑NY2 cells cultured at 37.5˚C were higher compared with those in cells cultured at 35.0˚C. In addition, adenosine‑5'‑triphosphate (ATP) levels in SRA01/04 and iHLEC‑NY2 cells were also significantly higher at 37.5˚C compared with those at 35.0˚C. By contrast, no significant differences in Na+/K+‑ATPase or Ca2+‑ATPase activities were observed between HLE cells cultured at 35.0 and 37.5˚C. These results suggested that expression of the mitochondrial genome was enhanced in high‑temperature culture, resulting in a sufficient ATP content and cell proliferation for lens opacity. Therefore, elevated environmental temperatures may increase the risk of nuclear cataracts caused by HLE cell proliferation via mitochondrial activation.

Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma. In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS), multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS is associated with an extremely poor prognosis, and effective treatment options are lacking. Using the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies using a human antibody phage library. We obtained antibody clones recognizing 95 independent antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing simple, rapid, and high-throughput selection of antibody clones. These results were highly correlated with those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with the antibody, the antigen-antibody complex was recovered with magnetic beads, and the protein bands from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS). CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies. In the future, antibody-drug conjugate targeting of these specific proteins may be promising for clinical applications.

Adipose-derived stem cell (ADSC) sheets have potential to be effective in various therapies. In this study, we first demonstrated that a cell sheet composed of human ADSCs could be created using a new temperature-responsive culture dish from the DIC Corporation. The dish can cause detachment of adherent cells due to temperature changes, but a few morphological analyses have evaluated the presence or absence of damage on the detached surface of cell sheet. To characterize our ADSC sheet, we tried to observe the surface of ADSC sheets with scanning electron microscope (SEM) using the ionic liquid, which enables the rapid preparation of samples. No damage was found on the surface of the ADSC sheets on the side that had been in contact with the surface of the culture dishes. In addition, when the transcriptomes of the harvested cell sheets were compared with those of monolayer cultures, no up-regulation of cell death related genes were detected. These results propose that the detachment from temperature-responsive culture dish causes no serious damage on the prepared ADSC sheet. It is also suggested that the SEM with ionic liquids is a useful and rapid method for the analysis of ADSC sheets for therapy.

When regenerated tissue is generated from induced pluripotent stem cells (iPSCs), it is necessary to track and identify the transplanted cells. Fluorescently-labeled iPSCs synthesize a fluorescent substance that is easily tracked. However, the expressed protein should not affect the original genome sequence or pluripotency. To solve this problem, we created a cell tool for basic research on iPSCs. Iris tissue-derived cells from GFP fluorescence-expressing mice (GFP-DBA/2 mice) were reprogrammed to generate GFP mouse iris-derived iPSCs (M-iris GFP iPSCs). M-iris GFP iPSCs expressed cell markers characteristic of iPSCs and showed pluripotency in differentiating into the three germ layers. In addition, when expressing GFP, the cells differentiated into functional recoverin- and calbindin-positive cells. Thus, this cell line will facilitate future studies on iPSCs.

Induced pluripotent stem (iPS) cells are widely used as a research tool in regenerative medicine and embryology. In studies related to lens regeneration in the eye, iPS cells have been reported to differentiate into lens epithelial cells (LECs); however, to the best of our knowledge, no study to date has described their formation of three-dimensional cell aggregates. Notably, in vivo studies in newts have revealed that iris cells in the eye can dedifferentiate into LECs and regenerate a new lens. Thus, as basic research on lens regeneration, the present study investigated the differentiation of human iris tissue-derived cells and human iris tissue-derived iPS cells into LECs and their formation of three-dimensional cell aggregates using a combination of two-dimensional culture, static suspension culture and rotational suspension culture. The results revealed that three-dimensional cell aggregates were formed and differentiated into LECs expressing αA-crystallin, a specific marker protein for LECs, suggesting that the cell-cell interaction facilitated by cell aggregation may have a critical role in enabling highly efficient differentiation of LECs. However, the present study was unable to achieve transparency in the cell aggregates; therefore, we aim to continue to investigate the degradation of organelles and other materials necessary to make the interior of the formed cell aggregates transparent. Furthermore, we aim to expand on our current work to study the regeneration of the lens and ciliary body as a whole in vitro, with the aim of being able to restore focusing function after cataract surgery.

It has recently been reported that lanosterol (LAN) plays a preventive role against lens opacification through the reversal of crystalline aggregation. However, the effect of LAN is not sufficient to restore lens transparency. In this study, we designed ophthalmic nanosuspensions (LAN-ONSs and NIL-ONSs) based on LAN and nilvadipine (NIL), which can counteract cataract-related factors (e.g., enhanced Ca2+ and calpain levels), and investigated whether the combination of LAN-ONSs and NIL-ONSs can restore the nuclear lens opacity in sodium-selenite-induced cataractic rats (cataractic rats). The mean particle sizes of the LAN-ONSs and NIL-ONSs were 108.8 nm and 89.0 nm, respectively. The instillation of the LAN-ONSs or NIL-ONSs successfully delivered the drugs (LAN or NIL) into the lenses of the rats, although the instillation of LAN-ONSs or NIL-ONSs alone did not increase lens transparency in the cataractic rats. On the other hand, the cataract-related factors (enhanced Ca2+ and calpain levels) were significantly alleviated by the combination of LAN-ONSs and NIL-ONSs; furthermore, the perinuclear refractile ring in the lens nucleus and enhanced number of swollen fibers were attenuated by the LAN-ONS and NIL-ONS combination. Moreover, the opacity levels in the cataractic rats were reduced after treatment with the combination of LAN-ONSs and NIL-ONSs. It is possible that the combination of LAN and NIL will be useful for the treatment of lens opacification in the future.

We previously found that 1% minoxidil (MXD) nanoparticles prepared using a bead mill method led to an increase I n hair follicle delivery and hair growth in C57BL/6 mice. In the present study, we designed a nanoparticle formulation containing 5% MXD (MXD-NPs) using the bead mill method and investigated the hair-growth effect of MXD-NPs and a commercially available MXD solution (CA-MXD). Hair growth and in vivo permeation studies were conducted using C57BL/6 mice. Moreover, we examined the MXD contents in the upper (hair bulge) and the lower hair follicle (hair bulb) and observed the hair follicle epithelial stem cells (HFSC) by immunohistochemical staining using the CD200 antibody. The mean particle size of the MXD in the MXD-NPs was 139.8 nm ± 8.9 nm. The hair-growth effect of the MXD-NPs was higher than that of CA-MXD, and the MXD content in the hair bulge of mice treated with MXD-NPs was 7.4-fold that of the mice treated with CA-MXD. In addition, the activation of HFSC was observed around the bulge in the MXD-NPs-treated mice. We showed that MXD-NPs enable the accumulation of MXD in the upper hair follicles more efficiently than CA-MXD, leading the activation of HFSC and the hair growth.

We previously reported that the bioavailability (BA) of irbesartan (IRB), a BSC class II drug, was improved by preparing nanocrystalline suspensions. However, nanocrystalline suspensions have chemical and physical instabilities and must be converted into tablets through drying approaches in order to overcome such instabilities. In this study, we attempted to design a molded tablet based on nanocrystalline IRB suspensions (IRB-NP tablet) and investigated the effects of this IRB-NP tablet on blood pressure (BP) in a stroke-prone spontaneously hypertensive (SHR-SP) rat. The IRB-NP tablet (with a hardness of 42.6 N) was developed by combining various additives (methylcellulose, 2-hydroxypropyl-β-cyclodextrin HPβCD, D-mannitol, polyvinylpyrrolidone, and gum arabic) followed by bead-milling and freeze-drying treatments. The mean particle size in the redispersions of the IRB-NP tablet was approximately 118 nm. The solubility and intestinal absorption of IRB in the IRB-NP tablet were significantly enhanced in comparison with the microcrystalline IRB tablet (IRB-MP tablet), and both solubility and clathrin-dependent endocytosis helped improve the low BA of the IRB. In addition, the BP-reducing effect of the IRB-NP tablet was significantly higher than that of the IRB-MP tablet. These results provide useful information for the preservation of nanocrystalline suspensions of BCS class II drugs, such as IRB.

Osteoarthritis (OA) is an irreversible degenerative condition causing bone deformation in the joints and articular cartilage degeneration with chronic pain and impaired movement. Adipose-derived stem cell (ADSC) or crushed adipose tissue injection into the joint cavity reportedly improve knee function and symptoms, including pain. Stem cell spheroids may be promising treatment options due to their anti-inflammatory and enhanced tissue regeneration/repair effects. Herein, to form human ADSC spheroids, we used first SphereRing® (Fukoku Co., Ltd., Ageo, Japan), a newly developed rotating donut-shaped tube and determined their characteristics by DNA microarray of mRNA analysis. The variable gene expression cluster was then identified and validated by RT-PCR. Gene expression fluctuations were observed, such as COL15A1 and ANGPTL2, related to vascular endothelial cells and angiogenesis, and TNC, involved in tissue formation. In addition, multiplex cytokine analysis in the medium revealed significant cytokines and growth factors production increase of IL-6, IL-10, etc. However, ADSC administration into the joint cavity involves their contact with the synovial fluid (SF). Therefore, we examined how SF collected from OA patient joint cavities affect 2D-culture ADSCs and ADSC spheroids and observed SF induced cell death. ADSC spheroids could become promising OA treatment options, although studying the administration methods and consider their interaction with SF is essential.

Posterior capsule opacification (PCO), the most common complication of cataract surgery occurring in 20-50% of patients after 2-5 years of cataract surgery, is a major problem in the aging society. The epithelial-mesenchymal transition (EMT) of lens epithelial cells after cataract surgery has been proposed as a major cause of PCO. Capsaicin, widely used as a food additive and analgesic agent, is a major pungent ingredient in red pepper. Although the effect of capsaicin on EMT has been reported in cancer cells, the biological reaction of capsaicin was unique in each cell type, and there have been no reports describing its effects on EMT earlier. In this study, we demonstrated that treatment with capsaicin inhibited TGFβ2-induced EMT in vitro lens epithelial cells and ex vivo explant lens epithelial cells. Furthermore, eye drops of capsaicin inhibited the PCO model mice in vivo. Finally, we showed that capsaicin inhibited non-canonically induced Smad2/3 activation via suppression of EGFR activation and ERK phosphorylation. Our findings indicate that capsaicin and its derivatives are good candidate compounds for preventing PCO after cataract surgery.

We developed ophthalmic formulations based on nilvadipine (NIL) nanocrystals (NIL-NP dispersions; mean particle size: 98 nm) by using bead mill treatment and investigated whether the instillation of NIL-NP dispersions delivers NIL to the lens and prevents lens opacification in hereditary cataractous Shumiya cataract rats (SCRs). Serious corneal stimulation was not detected in either human corneal epithelial cells or rats treated with NIL-NP dispersions. The NIL was directly delivered to the lens by the instillation of NIL-NP dispersions, and NIL content in the lenses of rats instilled with NIL-NP dispersions was significantly higher than that in the ophthalmic formulations based on NIL microcrystals (NIL-MP dispersions; mean particle size: 21 µm). Moreover, the supply of NIL prevented increases in Ca2+ content and calpain activity in the lenses of SCRs and delayed the onset of cataracts. In addition, the anti-cataract effect in the lens of rats instilled with NIL-NP dispersions was also significantly higher than that in NIL-MP dispersions. NIL-NPs could be used to prevent lens opacification.

We previously designed ophthalmic formulations (nTRA) containing tranilast nanoparticles (Tra-NPs) with high uptake into ocular tissues. In this study, we used in situ gel (ISG) bases comprising combinations of pluronic F127 (F127) and methylcellulose (MC/F127), pluronic F68 (F68/F127), and Carbopol (Car/F127), and we developed in situ gels incorporating Tra-NPs (Tra-NP-incorporated ISNGs) such as nTRA-F127, nTRA-MC/F127, nTRA-F68/F127, and nTRA-Car/F127. Moreover, we demonstrated the therapeutic effect on conjunctival inflammation using lipopolysaccharide-induced rats. Each Tra-NP-incorporated ISNG was prepared by the bead mill method, the particle size was 40-190 nm, and the tranilast release and diffusion from formulation were nTRA > nTRA-F127 > nTRA-F68/F127 > nTRA-Car/F127 > nTRA-MC/F127. In the Tra-NP-incorporated ISNGs, the tranilast residence time in the lacrimal fluid, cornea, and conjunctiva was prolonged, although the Cmax was attenuated in comparison with nTRA. On the other hand, no significant difference in conjunctival inflammation between non- and nTRA-F127-instilled rats was found; however, the nTRA-F68/F127, nTRA-Car/F127, and nTRA-MC/F127 (combination-ISG) attenuated the vessel leakage, nitric oxide, and tumor necrosis factor-α expression. In particular, nTRA-F68/F127 was significant in preventing the conjunctival inflammation. In conclusion, we found that the combination-ISG base prolonged the residence time of Tra-NPs; however, Tra-NP release from the formulation was attenuated, and the Tmax was delayed longer than that in nTRA. The balance of drug residence and diffusion in lacrimal fluid may be important in providing high ocular bioavailability in formulations containing solid nanoparticles.

We attempted to design irbesartan nanocrystalline (IRB-NC) suspensions by the bead mill method, and we evaluated the bioavailability (BA) in the oral administration of the nanocrystalline drug. The mean particle size of the IRB-NC suspensions was approximately 140 nm, and the crystalline structure of irbesartan in these suspensions was different using the bead mill method. The aggregation and degradation of irbesartan were not observed for one month, and the solubility increased. Moreover, the inclusion complex formation of IRB-NC suspensions with 2-hydroxypropyl-β-cyclodextrin was higher than that in traditional IRB powder (IRB-P). In addition, the intestinal absorption of IRB-NC suspensions was higher than that of IRB-P suspensions, and the reducing effect on blood pressure in spontaneously hypertensive SHR-SP rats orally administered IRB-NC suspensions was significantly higher than in those administered IRB-P suspensions. On the other hand, the intestinal penetration of IRB-NC suspensions was attenuated by the inhibitors of clathrin-dependent endocytosis (CME). In conclusion, we improved the low oral BA of irbesartan by preparing IRB-NC suspensions and showed that both the solubility and CME are related to the enhanced intestinal absorption of IRB-NC suspensions, resulting in an increase in their antihypertensive effect. These findings provide significant information for the development of oral nanomedicines.

BACKGROUND: Pneumothorax is one complication of transbronchial biopsy (TBB) using endobronchial ultrasonography with a guide sheath (EBUS-GS-TBB). We sought to clarify the risk factors for pneumothorax after EBUS-GS-TBB under fluoroscopic guidance. METHODS: We retrospectively reviewed data from 916 patients who underwent EBUS-GS-TBB at Fujita Health University Hospital. We evaluated the following risk factors for pneumothorax after EBUS-GS-TBB: patient characteristics (sex, age, and pulmonary comorbidities); lesion data (location, size, existence of ground-glass opacities [GGOs], pleural involvement, computed tomography [CT] bronchus sign, visibility on fluoroscopy, and EBUS findings); final diagnosis; years of bronchoscopist experience; and guide sheath size. Univariate and multivariate logistic regression analyses were performed. RESULTS: Among the 916 patients, 30 (3.28%) presented with pneumothorax. With a univariate analysis, factors that independently predisposed to pneumothorax included lesions containing GGOs, lesions in sagittal lung segments on fluoroscopy, lesions that were not visible on fluoroscopy, and infectious lesions. A univariate analysis also showed that lesions in the right upper lobe or left upper division, as well as malignant lesions, were less likely to lead to pneumothorax. Age, underlying pulmonary disease, CT bronchus sign, EBUS findings, bronchoscopist experience, and guide sheath size did not influence the incidence of pneumothorax. A multivariate analysis revealed that only lesions containing GGOs (odds ratio [OR] 6.47; 95% confidence interval [CI] 2.13-19.6, P = 0.001) and lesions in lung segments with a sagittal orientation on fluoroscopy (OR 2.47; 95% CI 1.09-5.58, P = 0.029) were significant risk factors for EBUS-GS-TBB-related pneumothorax. CONCLUSIONS: EBUS-GS-TBB of lesions containing GGOs or lesions located in sagittal lung segments on fluoroscopy correlate with a higher pneumothorax risk.

We previously designed a Carbopol gel formulation (N-IND/MEN) based on a combination of indomethacin solid nanoparticles (IND-NPs) and l-menthol, and we reported that the N-IND/MEN showed high transdermal penetration. However, the detailed mechanism for transdermal penetration of IND-NPs was not clearly defined. In this study, we investigated whether endocytosis in the skin tissue of rat and Göttingen minipig is related to the transdermal penetration of IND-NPs using pharmacological inhibitors of endocytosis. The pharmacological inhibitors used in this study are as follows: 54 µM nystatin, a caveolae-mediated endocytosis (CavME) inhibitor; 40 µM dynasore, a clathrin-mediated endocytosis (CME) inhibitor; and 2 µM rottlerin, a micropinocytosis (MP) inhibitor. The N-IND/MEN was prepared by a bead mill method, and the particle size of solid indomethacin was 79-216 nm. In both rat and Göttingen minipig skin, skin penetration of approximately 80% IND-NPs was limited by the stratum corneum (SC), although the penetration of SC was improved by the combination of l-menthol. On the other hand, the treatment of nystatin and dynasore decreased the transdermal penetration of indomethacin in rats and Göttingen minipigs treated with N-IND/MEN. Moreover, in addition to nystatin and dynasore, rottlerin attenuated the transdermal penetration of IND-NPs in the Göttingen minipigs' skin. In conclusion, we found that l-menthol enhanced the SC penetration of IND-NPs. In addition, this study suggests that the SC-passed IND-NPs are absorbed into the skin tissue by energy-dependent endocytosis (CavME, CME, and/or MP pathways) on the epidermis under the SC, resulting in an enhancement in transdermal penetration of IND-NPs. These findings provide significant information for the design of nanomedicines in transdermal formulations.

Cilostazol (CIL) exerted a protective effect by promoting blood-brain barrier integrity as well as improving the status of neurological dysfunctions following cerebral ischemia/reperfusion (I/R) injury. We attempted to design a 0.5% CIL carbopol gel using solid nanoparticles (CIL-Ngel), and then investigated the relationships between energy-dependent endocytosis and the skin penetration of CIL-Ngel in this study. In addition, we evaluated whether the CIL-Ngel attenuated I/R-induced brain injury in a middle cerebral artery occlusion (MCAO)/reperfusion model mouse. The particle size of CIL was decreased using a bead mill, and the CIL particles (14.9 × 1014 particles/0.3 g) in the CIL-Ngel were approximately 50-180 nm. The release of CIL in the CIL-Ngel was higher than that in gel containing CIL powder (CIL-Mgel), and the CIL particles were released from the CIL-Ngel as nanoparticles. In addition, the percutaneous absorption of CIL from the CIL-Ngel was higher in comparison with that from CIL-Mgel, and clathrin-dependent endocytosis and caveolae-dependent endocytosis were related to the enhanced skin penetration of CIL-NPs. In the traditional (oral administration of CIL powder, 3 mg/kg) and transdermal administration (CIL-Ngel, 0.3 g) for 3 days (once a day), the area under the plasma CIL concentration-time curves (AUC) was similar, although the CIL supplied to the blood by the CIL-Ngel was more sustained than that via oral administration of CIL powder. Furthermore, the CIL-Ngel attenuated the ischemic stroke. In conclusion, we designed a gel using solid CIL-NPs, and we showed that the sustained release of CIL by CIL-Ngel provided an effective treatment for ischemic stroke in MCAO/reperfusion model mice. These findings induce the possibilities of developing novel applications of CIL solid nanoparticles.

Regenerative medicine in ophthalmology that uses induced pluripotent stem cells (iPS) cells has been described, but those studies used iPS cells derived from fibroblasts. Here, we generated iPS cells derived from iris cells that develop from the same inner layer of the optic cup as the retina, to regenerate retinal nerves. We first identified cells positive for p75NTR, a marker of retinal tissue stem and progenitor cells, in human iris tissue. We then reprogrammed the cultured p75NTR-positive iris tissue stem/progenitor (H-iris stem/progenitor) cells to create iris-derived iPS (H-iris iPS) cells for the first time. These cells were positive for iPS cell markers and showed pluripotency to differentiate into three germ layers. When H-iris iPS cells were pre-differentiated into neural stem/progenitor cells, not all cells became positive for neural stem/progenitor and nerve cell markers. When these cells were pre-differentiated into neural stem/progenitor cells, sorted with p75NTR, and used as a medium for differentiating into retinal nerve cells, the cells differentiated into Recoverin-positive cells with electrophysiological functions. In a different medium, H-iris iPS cells differentiated into retinal ganglion cell marker-positive cells with electrophysiological functions. This is the first demonstration of H-iris iPS cells differentiating into retinal neurons that function physiologically as neurons.

The prevalence of nuclear cataracts was observed to be significantly higher among residents of tropical and subtropical regions compared to those of temperate and subarctic regions. We hypothesized that elevated environmental temperatures may pose a risk of nuclear cataract development. The results of our in silico simulation revealed that in temperate and tropical regions, the human lens temperature ranges from 35.0 °C to 37.5 °C depending on the environmental temperature. The medium temperature changes during the replacement regularly in the cell culture experiment were carefully monitored using a sensor connected to a thermometer and showed a decrease of 1.9 °C, 3.0 °C, 1.7 °C, and 0.1 °C, after 5 min when setting the temperature of the heat plate device at 35.0 °C, 37.5 °C, 40.0 °C, and 42.5 °C, respectively. In the newly created immortalized human lens epithelial cell line clone NY2 (iHLEC-NY2), the amounts of RNA synthesis of αA crystallin, protein expression, and amyloid β (Aβ)1-40 secreted into the medium were increased at the culture temperature of 37.5 °C compared to 35.0 °C. In short-term culture experiments, the secretion of Aβ1-40 observed in cataracts was increased at 37.5 °C compared to 35.0 °C, suggesting that the long-term exposure to a high-temperature environment may increase the risk of cataracts.

Recent epidemiological studies have hypothesized that the prevalence of cortical cataracts is closely related to ultraviolet radiation. However, the prevalence of nuclear cataracts is higher in elderly people in tropical areas than in temperate areas. The dominant factors inducing nuclear cataracts have been widely debated. In this study, the temperature increase in the lens due to exposure to ambient conditions was computationally quantified in subjects of 50-60 years of age in tropical and temperate areas, accounting for differences in thermoregulation. A thermoregulatory response model was extended to consider elderly people in tropical areas. The time course of lens temperature for different weather conditions in five cities in Asia was computed. The temperature was higher around the mid and posterior part of the lens, which coincides with the position of the nuclear cataract. The duration of higher temperatures in the lens varied, although the daily maximum temperatures were comparable. A strong correlation (adjusted R2 > 0.85) was observed between the prevalence of nuclear cataract and the computed cumulative thermal dose in the lens. We propose the use of a cumulative thermal dose to assess the prevalence of nuclear cataracts. Cumulative wet-bulb globe temperature, a new metric computed from weather data, would be useful for practical assessment in different cities.